Structural instability of recombinant DNA expression vectors results in DNA deletions and rearrangements that alter vector structure. This is a significant concern in large scale cultures grown to produce polypeptides encoded by these expression vectors. These vectors may be altered in a way that prevents expression of the encoded polypeptide. Thus, when the cultures are induced for expression of the polypeptide, a negative selective pressure toward a lack of polypeptide expression often results in an accumulation of the altered expression vectors.
In view of the above, regulatory agencies, such as the Food and Drug Administration, require full characterization of any recombinant DNA expression vectors that are utilized to produce heterologous proteins. Evidence must be submitted to verify that the recombinant DNA expression vector is the same at the end of the fermentation as the expression vector from the original inoculum. Certification data includes structural and size analysis of the expression, and verification of the nucleotide sequence that codes for the desired product, and the regions flanking this coding sequence, especially flanking sequences that perform important functions, such as promoters.
Recombinant DNA vectors which utilize the Escherichia coli bacteriophage lambda pL promoter-operator region to enable transcription of an operably linked gene are often plagued by structural instability. When such vectors are examined at the end of the fermentation process, the structure of the vectors is often altered. It is the purpose of the present invention to provide transcriptional activating sequences that enhance the stability of the expression vectors while providing regulatable transcription of an operably linked gene.
For the purpose of the present invention, as disclosed and claimed herein, the following terms are as defined below:
ApR--the ampicillin resistant phenotype or gene conferring the same. PA1 cI857--the gene encoding a temperature sensitive form of the bacteriophage lambda cI repressor. PA1 EK-Bovine Growth Hormone--methionyl-phenylalanyl-(aspartyl).sub.4 -lysinyl-bovine growth hormone, with indicated amino-terminal amino acid sequence phenylalanyl-(aspartyl)-.sub.4 -lysinyl specifying an enterokinase cleavage site. PA1 EK-BGH--EK-Bovine Growth Hormone PA1 Functional Polypeptide--A recoverable, biologically active heterologous or homologous polypeptide or precursor, a recoverable bioactive polypeptide comprised of a heterologous polypeptide and a partial or whole homologous polypeptide, or a recoverable bioinactive polypeptide containing a heterologous polypeptide and a bio-inactivating polypeptide which can be specifically cleaved or a bioinactive polypeptide that can be converted to a bioactive polypeptide by refolding or other chemical treatments known to those skilled in the art. PA1 pL--bacteriophage lambda leftward promoter. PA1 Promoter--a DNA sequence that directs transcription of DNA into RNA. PA1 Recombinant DNA Expression Vector--any recombinant DNA cloning vector into which a promoter has been incorporated. PA1 Recombinant DNA Cloning Vector--any agent including, but not limited to, recombinant plasmids, bacteriophages, and viruses, consisting of a DNA molecule to which one or more additional DNA segments can or have been added. PA1 Structural Gene--any DNA sequence that encodes a functional polypeptide, inclusive of that DNA encoding the start and stop codons. PA1 TetR--the tetracycline resistant phenotype or gene conferring the same. PA1 Transcriptional Activating Sequence--a DNA sequence that directs the transcription of DNA in a regulatable fashion. PA1 Translational Activating Sequence--any DNA sequence, including the ribosome binding site and translational start codon, such as 5'-ATG-3', but not including any sequences downstream from the start codon, that provides for the initiation of translation of a mRNA transcript into a peptide or polypeptide.